P-Glycoprotein (Pgp) is a plasma membrane protein, which functions as an efflux pump to transport multiple cytotoxic drugs out of the cell utilizing energy from ATP hydrolysis. Pgp is responsible for most cases of multidrug resistance (MDR) in tumors and thus limits the success of chemotherapy. Pgp has been shown to have high basal and drug-stimulated ATPase activity. Recent studies have shown that Pgp may have several functional states during each cycle of ATP binding, hydrolysis, and drug transport. The general goal of this application is to understand the molecular mechanism of Pgp-mediated drug transport and its relationship to the topological folding of Pgp. To achieve this general goal, we plan to accomplish the following specific aims: (1) to investigate the structural change of Pgp in its catalytic cycle; (2), to investigate the structural change of Pgp elicited by drug binding; (3) to investigate the involvement of topological change of Pgp in its function; and (4) to investigate the molecular mechanism by which ribosomes and ribosome- associated proteins determine the topological folding of Pgp. The information and probes obtained from this study will help us understand the molecular mechanism of Pgp-mediated drug transport, the relationship between the topological folding and function of Pgp, and the molecular basis of Pgp-mediated MDR. It thus will help increase the effectiveness of cancer chemotherapy. Results from this study will also help us understand how the membrane folding of mammalian polytopic membrane proteins is regulated in general. This work may lead us to the discovery of a new family of chaperone proteins associated with ribosomes that are important in determining the folding of membrane proteins co-translationally.